Probe for diagnosing infectious diseases

ABSTRACT

A probe derived from bacteria of pneumonia, containing fragments of DNA which  streptococcus pneumoniae  essentially possesses, and useful for detecting and identifying causative bacteria of pneumonia is obtained by completely digesting the DNA with a restriction endonuclease PstI, followed by cloning into a suitable vector.

This application is the national stage of International Application No.PCT/JP95/02036, filed Oct. 2, 1995.

FIELD OF THE ART

The present invention relates to a probe which is useful for detectingand identifying the causative bacteria of infectious diseases,especially Streptococcus pneumoniae which is a representative causativebacterium of bacterial pneumonia.

BACK GROUND ART

In pathology, infection is defined as an invasion and an establishmentof an anchorage for growth in a host organism by a pathogenic organism(hereinafter referred to as “bacterium”). The outbreak of a diseasecaused by proliferation of a bacterium in vivo depends upon theinterrelationship between the resistance of the host organism and thevirulence of the bacterium.

To improve therapeutic systems for treatment of infectious diseases,especially inflammatory diseases caused by Streptococcus pneumoniae andthe like in pulmonary lobes and bronchia, namely bacterial pneumonia,among the infectious diseases, has been urgent problem in the field ofthis art. Such bacterial pneumonia is triggered by an attack withbacteria, e.g., Streptococcus pneumoniae, Staphylococcus aureus and thelike which cause inflammation predominantly in alveoli. When sufferedfrom bacterial pneumonia, generally from clinical aspect, inflammatorysymptoms in upper airway followed by sudden chill and shiver attach,then crisis of high fever around 40° C., and terrible cough, stethalgiaand sputum have appeared.

Further, the bacterial pneumonia is a serious and exigent infectiousdiseases in which severe systemic symptoms such as malaise, anorexia andheadache have involved, presenting dyspnea, cyanosis, and could beaccompanied by bacteremia, cerebrospinal meningitis or arthritis as thecomplication thereof, finally could sometimes lead to a lethal process.

Thus, improvement in rapid method for therapy of bacterial pneumonia hasbeen awaited since appropriate therapy to be put into practice in earlystage based on accurate and quick diagnosis is necessary.

Moreover, when suffered from an infectious disease including pneumonia,it has been believed that phagocytes including neutrophils, monocytesand macrophages primarily function in defense of the body, and thatexuded bacteria from the texture of the phagocyte which hadpredominantly grown have appeared into blood.

In general, bacterial pneumonia is defined as a case wherein the abilityof phagocytosis by cells cannot overcome the virulence of the bacteriaand then the bacteria such as Streptococcus pneumoniae settle on thepulmonary lobe and the tissue of bronchia to cause inflammation. Inconventional method for diagnosing bacterial pneumonia, the followingitems should be checked: 1) clinical symptoms; 2) culture of a specimen;3) gram-staining of the bacteria contained in the specimen; and 4) shockstate. After those items have been clarified, the course of therapy hasbeen oriented. In its typical case, the above mentioned clinicalsymptoms, stethendoscopic findings, and increase in neutorophils,increase in acute phase response substances such as CRP (C-ReactiveProtein) make speculation of diagnosis possible, however, for definitivediagnosis, the causative bacteria must be searched and determined fromthe specimen such as sputum, hydrothorax or blood, and then treatmentmust be conducted using proper antibiotics responding to the species ofthe bacteria. Accordingly, rapid and reliable identification of thecausative bacteria has been awaited in the clinical site.

Additionally, novel types of pneumonia e.g., Legionellosis andPneumocystis carinii pneumonia are identified, and resistant strainssuch as MRSA (methicillin-resistant Staphylococcus aureus) have beenappeared recently, the importance in searching the causative bacteriahave been growing.

However, as a matter of fact, difficulties have usually accompanied inconfirming the causative bacteria. Especially, in case of communityacquired pneumonia, it is known that therapy is initiated in 30-50% ofthe cases under such circumstances wherein the causative bacteriathereof are not clarified yet. As a method for identifying the causativebacteria in a patient who is suspected to be suffered from bacterialpneumonia, the following common procedures are adopted: employing thesample collected from sputum, secretion from upper airway, hydrothorax,topical focus, or blood as a specimen to estimate applicability of thesample as a test material by observing inflammatory cells of smearthereof, then, determining cell type by Gram staining e.g., gramnegative or positive, and coccus or bacillus, and finally, culturing thebacteria using selection medium to identify the causative bacteria.

In accordance with this method, however, culturing the bacteria takeslong time, and contamination of indigenous bacteria could not be avoidedas well. Otherwise, in case that a lot of antibiotics have beenadministered when bacterial pneumonia had been suspected, even thoughbacteria are contained in the specimen, proliferation or growth wouldoften be prevented, thus the rate of success in culturing the bacteriafrom the specimen has become actually quite low.

Although available subroutine methods including instrumental analysismethod of constituents of bacteria and metabolic products by bacteria(See Yoshimi Benno, “Quick identification of bacteria with gaschromatography”, Rinsho Kensa, Vol. 29, No.12, 1618-1623, November 1985,Igaku Shoin.), a method utilizing a specific antibody (See, JapanesePatent Provisional Publication No.60-224068.), and a hybridizationmethod utilizing a specificity of DNA (Japanese Phase Patent ProvisionalPublication No. 61-502376) have been developed, any of which requiresseparation, culturing and growing of the bacteria.

On the other hand, as an established method based on the function of thephagocyte in infectious diseases, there is a method to examine a stainedsmear of buffy coat wherein leukocytes in the blood sample areconcentrated, under an optical microscope. Generally speaking, the rateof detection of bacteria in buffy coat specimens from adult bacteremiapatients is 30% at most, which is similar to that in blood specimensfrom an earlobe. However, it was reported that in case that the patientswere newborn children, bacteria had been detected in seven cases in tencases total (70%). Therefore, information concerning the presence ofbacteria in peripheral blood obtained by a microscopic examination on asmear is an important index for therapy.

Since the above mentioned conventional methods necessitate thepretreatment which requires at least three to four days in totalincluding one to two days for selective isolation of bacteria from aspecimen, one day for culture, and one or more days for operation offixation, and then the culture thereof is continued in practice untilthe bacteria grow. Therefore, in many cases, the pretreatment requiresone week or more. Furthermore, there has been a risk on contamination ofa other bacteria which could not be distinguished from the causativebacteria during the culture period.

As an important matter, under such circumstances above described, thenumber of bacteria that can be grown is small even under appropriateconditions for culture, because many bacteria in a specimen to be grownhave been ingested into phagocyte, dead or on a static state due toantibiotics administered. Therefore, the actual detection rate ofbacteria is as low as about 10% when the clinical specimen culturemethod is employed. In the other words, for the present, the presence ofbacteria in 90% of the examined blood from the patient suspectedclinically as suffering from pneumonia, which has been cultured forfurther one or more days, could not be proved after all.

Thus, in light of the situation above, the present practice depends on atrial and error treatment method, starting when pneumonia is clinicallysuspected without awaiting the detection results of the identification,wherein an antibiotic having the effectiveness for the widest range ofthe causative bacteria is administered first while the causativebacteria is still unknown, and if the antibiotic is not effective afterone or two days, then another antibiotic will be tested, regardless ofthe fact that determination of the causative bacteria and selection ofthe suitable antibiotics are required.

According to the method to stain the bacteria in a specimen, theconstituents of the living body are likewise stained together withbacteria, therefore, a skilled experience to identify bacteria quicklyaccording to their image through a microscope is required, then theremay be cases that can be hardly identified.

Although pneumonia is a disease wherein a rapid and exact diagnosis hasbeen required as stated above, the conventional diagnosis method couldnot have satisfied such requirements.

SUMMARY OF THE INVENTION

The present invention was accomplished in view of the above-describedproblem in the art, and according to one aspect of this invention, thereis provided a probe having a specific reactivity with DNA or RNAobtained from causative bacteria of the infectious diseases, especially,Streptococcus pneumoniae which is the representative causative bacteriaof bacterial pneumonia, and nucleotide sequences of a portion of thegene essentially included in Streptococcus pneumoniae being elucidated.

Namely, the probe of the present invention allows significant detectionof remaining bacterial DNA, the bacteria being incorporated intophagocytes and destroyed, thereby quick and accurate detection method ofthe causative bacteria of the infectious diseases would be availablewithout culture and/or growth of the bacteria. Moreover, when a primeris designed with reference to information on the nucleotide sequence ofthe probe, causative bacteria can be identified without hybridizationstep, through amplifying DNA by means of a PCR technique.

Additionally, when a non-radioactive probe, e.g., a biotinylated probe,is used for hybridization step, detection in a general laboratory can beperformed as well using an optical microscope, and the detection processwill be carried out rapidly and simply.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates restriction enzyme maps of probes SP-22 (SEQ ID NO:1), SP-23 (SEQ ID NO: 2), and SP-25 (SEQ ID NO: 3) for detectingStreptococcus pneumoniae; and

FIG. 2 illustrates restriction enzyme maps of probes SP-26 (SEQ ID NO:4), SP5-15 (SEQ ID NO: 5), SP5-34 (SEQ ID NO: 6), and SP6-6 (SEQ ID NO:7) for detecting Streptococcus pneumoniae.

BEST MODE FOR CARRYING OUT THE INVENTION

Examples of the probes from Streptococcus pneumoniae which is acausative bacterium of infectious diseases, especially of pneumonia aredescribed below:

EXAMPLE 1 DNA Probe from Streptococcus pneumoniae

(1) Preparation of DNA Probe from Streptococcus pneumoniae

Clinical isolate of Streptococcus pneumoniae was cultured overnight inBHI (Brain Heart Infusion) medium, then bacteria from the culture werecollected, added thereto N-Acetylmuramidase SG. Thereafter genomic DNAwas extracted according to Saito-Miura's Method (“Preparation ofTransforming Deoxyribonucleic Acid by Phenol Treatment”, Biochim.Biophys. Acta, Vol.72, pp.619-629(1963)).

The extracted DNA was digested completely with restriction enzyme PstIand random cloned into vector pGEM-3Z. Among thus obtained clones, sevenprobes unique to the bacteria Streptococcus pneumoniae, namely probescomprising DNA fragment showing specificity to DNA from Streptococcuspneumoniae were then selected.

The selected probes were then designated as probe SP-22, probe SP-23,probe SP-25, probe SP-26, probe SP-5-15, probe SP 5-34 and probe SP-6-6(SEQ ID NOS: 1-7, respectively), and restrictions maps of which areillustrated in FIG. 1 and FIG. 2.

Reactivities between each probe and DNA from several kinds of causativebacteria of infectious diseases were examined according to the followingmethod.

First, as subject strains for an examination, strains of clinicalisolate and deposited strains described below in Table 1 were prepared.Human genomic DNA and control sample in Table 1 were obtained andprepared from leukocyte collected from four healthy adult men, andEscherichia coli K-12 JM109 transformant comprising plasmid pGem-32,respectively.

TABLE 1 bacterium No. strain name original source 1 Streptococcuspneumoniae NYSDH DP-2 2 Streptococcus pneumoniae clinical isolate 3Streptococcus pneumoniae clinical isolate 4 Streptococcus agalactiae IFM58/59 5 Streptococcus anginosus NCTC 8787 6 Streptococcus constellatusATCC 27823 7 Streptococcus equisimilis NCTC 8543 8 Streptococcus faeciumNCTC 7171 9 Streptococcus faecalis ATCC 19433 10 Streptococcus mitisATCC 9811 11 Streptococcus morbillorum ATCC 27824 12 Streptococcuspyogenes DHI S8 13 Streptococcus sanguis ATCC 10556 14 Streptococcussalivarius ATCC 7073 15 Staphylococcus aureus ATCC 25923 16Staphylococcus epidermidis ATCC 12228 17 Escherichia coli ATCC 25922 18Klebsiella pneumoniae clinical isolate 19 Pseudomonas aeruginosa ATCC27583 20 Enterococcus agglomerans clinical isolate 21 Haemophilisinfluenzae clinical isolate 22 Candida albicans IFM 40083-A type 23Aspergillus fumigatus MTU 06001 24 Cryptococcus neoformans MTU 13001 25Mucor spinosus TIMM 1322 26 human genomic DNA 27 control [note] NYSDH ;New York State Department of Health (Albany, New York, U.S.A.) NCTC ;National Collection of Type Cultures (London, England) DHI ; DairenHygienic Institute IFM ; Chiba University, Eucaryotic MicroorganismResearch Center MTU ; Tokyo University, Medical Faculty TIMM ; TeikyoUniversity, Medical Fungus Research Center

Then, DNA of each strain was extracted according to the method of theabove Example 1 (1), and samples for dot-blot-hybridization wereobtained by spotting certain amount (e.g., 5 μl) of the extracted DNA toa nylon filter and then conducting alkaline denaturation.

Human genomic DNA sample was prepared from the above-described leukocyteemploying Saito-Miura's method (supra). Meanwhile, control sample wasprepared from the above-described Escherichia coli K-12 JM109transformant comprising plasmid pGem-32 applying the method forpreparing plasmid DNA described in Example 2 (1) below. Hybridization onDNA probes from Streptococcus pneumoniae labeled withDigoxigenin-11-dUTP (BRL) was then performed overnight according toManual of Maniatis (T. Maniatis, et al., “Molecular Cloning (ALaboratory Manual)”, Cold Spring Harbour Laboratory (1982)), under thecondition of 45% formamide, 5×SSC, 42° C.

Samples obtained by overnight hybridization were washed twice with0.1×SSC, 0.1% SDS for 20 minutes at 55° C., then, hybridization wasevaluated by detection through color reaction using Anti-Dig-ALPconjugates (BRL). Experimental results on hybridization between each ofthe probes and DNAs from each clinical isolate are summarized in Table 2below.

TABLE 2 probe [denotation: SP−] No. bacterium strain name (origin) 22 2325 26 5-15 5-34 6—6  1 Streptococcus pneumoniae (NYSDHDP-2) + + + + + + +  2 Streptococcus pneumoniae (clinicalisolate) + + + + + + +  3 Streptococcus pneumoniae (clinicalisolate) + + + + + + +  4 Streptococcus agalactiae (IFM 58/59) − − − − −− −  5 Streptococcus arginosus (NCTC 8787) − − − − − − −  6Streptococcus constellatus (ATCC 27823) − − − − − − −  7 Streptococcusequisimilis (NCTC 8543) − − − − − − −  8 Streptococcus faecium (NCTC7171) − − − − − − −  9 Streptococcus faecalis (ATCC 19433) − − − − − − −10 Streptococcus mitis (ATCC 9811) − − − − − − − 11 Streptococcusmorbillorum (ATCC 27824) − − − − − − − 12 Streptococcus pyogenes (DHIS8) − − − − − − − 13 Streptococcus sanguis (ATCC 10556) − − − − − − − 14Streptococcus salivarius (ATCC 7073) − − − − − − − 15 Staphylococcusaureus (ATCC 25923) − − − − − − − 16 Staphylococcus epidermidis (ATCC12228) − − − − − − − 17 Escherichia coli (ATCC 25922) − − − − − − − 18Klebsiella pneumoniae (clinical isolate) − − − − − − − 19 Pseudomonasaeruginosa (ATCC 27583) − − − − − − − 20 Enterococcus agglomerans(clinical isolate) − − − − − − − 21 Haemophilis influenzae (clinicalisolate) − − − − − − − 22 Candida albicans (IFM 40083-A type) − − − − −− − 23 Aspergillus fumigatus (MTU 06001) − − − − − − − 24 Cryptococcusneoformans (MTU 13001) − − − − − − − 25 Mucor spinosus (TIMM 1322) − − −− − − − 26 human genomic DNA − − − − − − − 27 control + + + + + + +[note] “+” denotes that the hybridization signal was detected, “+”denotes that the hybridization signal was not detected, respectively.

Apparently from Table 2 above, any of the probes exhibited reactivityspecifically with only DNA obtained from Streptococcus pneumoniae, anddid not exhibit cross-reactivity (ability to hybridize) with any DNAobtained from bacteria other than genus Streptococcus, as well as DNAfrom other species of Streptococcus, thus specificity thereof to thespecies Streptococcus pneumoniae has been confirmed.

EXAMPLE 2 Analysis of Nucleotide Sequence

Nucleotide sequences of DNA probes (7 probes total) of which specificityto the bacterial species were verified in the Example 1, were determinedaccording to the following method.

(1) Preparation of Plasmid DNA

Escherichia coli K-12, JM109 transformant, comprising the subclonedinsert fragment (to be sequenced) in pGem-3Z (Promega), was inoculatedin 5 ml of Luria-Bactani Medium (bacto-tryptone, 10 g/1 L; bacto-yeastextract, 5 g/1 L; NaCl, 10 g/1 L; adjusted pH to 7.0 with 5N NaOH) andcultured overnight.

Culture medium was centrifuged (5,000 rpm, 5 min.) to collect thebacterial cell body. One hundred μl of a solution of 50 mM glucose/50 mMTris-HCl (pH8.0)/10 mM EDTA containing 2.5 mg/ml of lysozyme (Sigma) wasadded to the precipitate, and left at room temperature for 5 minutes. Tothe suspension thus obtained, 0.2M NaOH solution containing 1% of sodiumdodecyl sulfate (Sigma) was added and mixed therewith. One hundred andfifty μl of 5M potassium acetate aqueous solution (pH. 4.8) was furtheradded thereto and mixed, then cooled on ice for 15 minutes.

The supernatant obtained by centrifugation (15,000 rpm, 15 min.) of themixture was treated with phenol/CHCl₃ and added thereto ethanol of twotimes by volume, then the precipitate was obtained by furthercentrifugation (12,000 rpm, 5 min.). This precipitate was dissolved in100 μl of solution of 10 mM Tris-HCl (pH7.5)/0.1 mM EDTA, and addedthereto 10 mg/ml RNaseA (Sigma) solution, then left it at roomtemperature for 15 minutes.

Three hundred μl of 0.1M sodium acetate aqueous solution (pH 4.8) wasadded to this preparation and treated with phenol/CHCl₃, then theprecipitate was obtained therefrom by adding ethanol to the supernatant.This precipitate was dried and dissolved in 10 μl of distilled water togive a DNA sample.

(2) Pretreatment for Sequencing

Pretreatment for sequencing was performed with AutoRead (TM) SequencingKit (Pharmacia).

Briefly, concentration of DNA for use as a template was adjusted to 5-10ug in 32 μl of solution. Thirty two μl of the template DNA solution wastransferred to 1.5 ml mini-tube (Eppendolf), and added thereto 8 μl of2M NaOH aqueous solution, then mixed gently. After instantcentrifugation, it was left at room temperature for 10 minutes.

Seven μl of 3M sodium acetate (pH 4.8) and 4 μl of distilled water wereadded thereto, then 120 μl of ethanol was also added and mixed, and themixture was left for 15 minutes on dry ice. Then, DNA which wasprecipitated by centrifugation for 15 minutes was collected, and thesupernatant was removed carefully therefrom. The precipitate obtainedwas washed with 70% ethanol and centrifuged for 10 minutes. Then, afterthe supernatant was removed carefully again, the precipitate was driedunder the reduced pressure.

The precipitate was dissolved in 10 μl of distilled water, then 2 μl offluorescent primer (0.42 A₂₆₀ unit/ml, 4-6 pmol), (Fluorescent Primer,M13 Universal Primer; 5′-Fluoroscein-d[CGACGTTGTAAAACGACGGCCAGT(SEQ IDNO: 8)]-3′ (1.6 pmol/μl; 0.42 A₂₆₀ unit/ml); M13 Reverse Primer,5′-Fluoroscein-d[CAGGAAACAGCTATGAC(SEQ ID NO: 9)]-3′ (2.1 pmol/μl; 0.42A₂₆₀ unit/ml)) and 2 μL of buffer for annealing were added thereto, andmixed gently.

After instant centrifugation, the mixture was heat-treated at 65° C. for5 minutes and rapidly transferred to a circumstance of 37° C. and keptthe temperature for 10 minutes. After keeping the temperature, it wasleft at room temperature for 10 minutes or more and centrifugedinstantly. Then, a sample was prepared by adding 1 μl of a buffer forelongation and 3 μl of dimethyl sulfoxide thereto.

Four mini-tubes were identified with any of the marks of “A”, “C”, “G”or “T”, and, according to each of the mark, respective 2.5 μl of A Mix(dissolved ddATP with dATP, dCTP, c⁷dGTP and dTTP), C Mix (dissolvedddCTP with dATP, dCTP, c⁷dGTP and dTTP), G Mix (dissolved ddGTP withdATP, dCTP, c⁷dGTP and dTTP), and T Mix (dissolved ddTTP with dATP,dCTP, c⁷dGTP and dTTP) were poured into each identified tube.

Each solution was preserved on ice until it was used, and was kept at37° C. for one minute or more before use.

Two μl of diluted T7DNA polymerase (Pharmacia; 6-8 units/2 μl) was addedto the DNA sample, and completely mixed through pipetting or mixing itgently. Immediately after the mixing was completed, the mixed solutionwas dispensed to 4.5 μl of the above four different solutionrespectively which had been thermal controlled. Fresh tip was used ateach time of dispensation.

The solution was kept for 5 minutes at 37° C., then 5 μl of solution forterminating reaction was added to each reaction solution. Fresh tipswere also used for dispensation on this dispensation step. Immediatelyafter keeping the solution for 2-3 minutes at 90° C., it was cooled onice. For electrophoresis, 4-6 μl of the solution per one lane wasapplied.

(3) Sequencing on Nucleotide Sequence

Sequencing on each nucleotide sequences of the probes disclosed inExample 1 and 2 having specificity to DNA from Streptococcus pneumoniaewas performed using A.L.F. DNA Sequencer System (Pharmacia) under anelectrophoresis condition of 45° C. for 6 hours.

Consequently, the entire nucleotide sequences of each of the probes:probe SP-22 (SEQ ID NO:1); probe SP-23 (SEQ ID NO:2); probe SP-25 (SEQID NO:3); probe SP-26 (SEQ ID NO:4); probe SP-5-15 (SEQ ID NO:5); probeSP-5-34 (SEQ ID NO:6); and probe SP-6-6 (SEQ ID NO:7) were clarified.

INDUSTRIAL APPLICABILITY

According to a probe of the present invention, bacteria for example,causative bacteria ingested into phagocytes can be directly detectedwithout proliferating the bacteria, and can be identified rapidly andaccurately. That is to say, according to diagnosis method using a probeof the present invention, identification of the bacteria can beaccomplished with single specimen, then, the necessary time fordiagnosis can be greatly reduced to about one to two days, while theconventional method (with low detection rate) required 3-4 days, and thedetection efficacy is remarkably improved. Therefore, the presentinvention can provide breakthrough guide for the treatment of bacterialpneumonia, then enable the effective treatment in the early stage of theinfectious diseases to the patient, as well as contribute to reductionin the mortality thereby.

Moreover, by clarifying the nucleotide sequences of the probes whichspecifically react with DNA from Streptococcus pneumoniae, one of themost closely related bacteria to attack of pneumonia among the causativebacteria of infectious diseases, artificial preparation of these probescould also be realized. Using the primers prepared by utilizing theinformation of the nucleotide sequences presently analyzed, DNA from thecausative bacteria contained in the clinical specimen can be amplifiedby PCR technique, to serve rapid diagnosis of causative bacteria.

Further, by comparing the nucleotide sequences of genomic DNA from theclinical specimen with those analyzed in accordance with the presentinvention, rapid identification of the species of the causative bacteriaof pneumonia can be carried out.

As stated above, the present invention provides a desired probe fordiagnosing the infectious diseases, besides, excellent utilities areexpected as a guide for preparing PCR primers and as a standard sequencesuitable for the comparison and reference with genomic DNA from theclinical specimen. Additionally, the present invention exerts furtheroutstanding effects, for example, of providing valuable clues forpreparation and development of the other probes which specifically reactwith DNA from causative bacteria of the infectious diseases (causativebacteria of pneumonia).

Moreover, since the nucleotide sequences disclosed in the presentapplication was obtained by random cloning of the genomic DNA fromclinical isolates, thus, utilities of the nucleotide sequences disclosedin the present invention should be extended to the complementarysequences thereof.

Further, although it would be assumed that DNA obtained from the wildstrain might contain a mutated portion, apparently from the disclosureof the examples above, such mutated DNA portion would never affect theutilities to be derived from the present invention such as thespecificity of the probe of the present invention upon use forhybridization for diagnosis of the infectious diseases, and usage of theinformation on the nucleotide sequences disclosed in the presentapplication to design the primer for PCR technique with the aim of arapid diagnosis of the infectious diseases.

9 1 368 DNA Artificial sequence Synthetic probe 1 ctgcagcttt caaggaacctgtcaagagag ccagtttcaa attgggaaaa aggttctgta 60 aactctcaaa gtgttgctctgcgaggattt ctgttggtac cattagggca gcctgataac 120 ctgctgtcac tgccgcaaacatggccaagc cagcgactac cgttttttcc actccccaca 180 tccccttgta ggagacgattcatgtggtgg tcggacttca tatcagttaa aatttcctgc 240 aaactctttt cctgagcttgggtcagggca aaaggaagac ttactttaac tgctgtcact 300 ttttcctgag accaattcagaaccagacca cttccctgaa ctctattttc agacttgagc 360 atctgcag 368 2 1978 DNAArtificial sequence Synthetic probe 2 ctgcaggttc ccctgtattt gctggtttcattactggttt aatcatggga gatgtgacta 60 ctggtttact tatcggtggt aacttgcaactgttcgttct tggggttggt accttcggtg 120 gtgcttctcg tatcgacgca acttctggtgcggttcttgc gacagccttc tctgtttcac 180 aaggaattga tgcaccgctt gccattactacaatcgctgt accagtagca gctctcttga 240 cttacttcga cgttcttggt cgtatgactactaccttctt ngctcaccgt gtggatgctg 300 caatcgaacg ctttgactat aaggtattgaacgcaactac ttgcttggtg cgattcgtgg 360 gctctatctc gtgcccttcc agtcttctttgcccttgctt ttggtggtgc ctttgtacaa 420 tcagtagtag acttcgttga agcctacaaatgggttgcat atggcttgac acttgcagga 480 cgtatgcttc caggtcttgg atttgcaatcttgcttcgtt accttccagt taaacgtaac 540 cttcactacc ttgctatggg atttggtttgacagctatgt tgactgttct ttactcatat 600 gtaacaggtc ttggtggcgc tgttgctggtatcgtaggta ctcttcctgc tgaagttgct 660 gaaaaaattg gtttcgtgaa caacttcaaaggtttgtcta tgattggtat ttctatcgta 720 ggtattttcc ttgcagtgct tcacttcaaaaatagccaaa aagtagctgt agcagcacct 780 tctacaccat cagaaagtgg ggaaatcgaagatgacgaat tctaattaca aacttacaaa 840 agaagatttt aatcaaatca acaaacgtagcttgtttact ttccaattag gttggaacta 900 cgaacgtatg caagcttctg gttacctttacatgatcttg cctcagttgc gtaaaatgta 960 tggtgatgga actcctgaat tgaaagaaatgatgaaagtt catactcaat tcttcaatac 1020 ttcaccattc ttccatacca ttatcgctggttttgacctt gccatggaag aaaaagatgg 1080 tgtaggttca aaagacgccg ttaacggtatcaagacaggt ttgatgggac cattcgctcc 1140 tcttggggat acaatctttg cttcacttgtacctgctatc atggggtcag tcgcagcaac 1200 tatggctatc gctggccaac cttgggggatcttcctttgg attgcagttg cagtagcgta 1260 tgacatcttc cgttggaaac agttggaatttgcttacaaa gaaggggtta accttatcaa 1320 caacatgcaa agtaccttga cagctttgattgacgctgca tctgtacttg gtgtcttcat 1380 gatgggtgct cttgtagcaa cagtgattaactttgaaatt tcttacaagt tgccaatcgg 1440 tgaaaagatg attgatttcc aagacatcttgaatcaaatc ttcccacgtt tgcttccagc 1500 aatctttact gcctttatct tctggttgcttggtaagaaa ggtatgaact ctactaaagc 1560 tatcggtatt attatcgtac ttgctttggctctttctgcc cttggtcact ttgcacttgg 1620 aatgtaattc cttatgacta aatcattaattttgtgagcc atggtcgctt ctgtgaggag 1680 cttagaggta gcacagaaat gattatgggcccacaagaca acatttacac agtagctctt 1740 cttccagaag atggcccaga agaatttactgctaaatttg aagctgttat tggaggattg 1800 gatgatttcc tagtctttgc ggatcttctcggtgggacac cttgtaatgt ggtgagtcgc 1860 ttgatcatgg aaggtcgtga tattgacctttacgcaggga tgaatcttcc aatggtgatt 1920 gaatttatca atgcgagcct tacaggcgcagatgcggact acaagagccg tgctgcag 1978 3 1124 DNA Artificial sequenceSynthetic probe 3 ctgcaggttt tgtcctcaac ctcccaatca aagaaaatat gagaaatctgcgagttaaga 60 ttgagaaaaa gacgggccta ctatggaata gatggcaaac aatctatgaaaacagaccaa 120 ttttagctca accccaccgt aaaattaccc attggggtac gacattgaattccaaggtga 180 gtgacgatga tgtcttgtaa tctgatggta gaatgacagt tagtttgtctagtttataag 240 aaagtactac ctgagcttga atagaactca ggtagctctc tatgaaagaacaaaattaat 300 actcaatgaa aatcaaagag caaactagga aactagccgc agtttgctcaaagcactgct 360 ttgaggttgt agataagact gacgaagtcg tcaccatata taatccaaggcgacgttgac 420 gtggattgaa gagattttag aagagtataa acagaaaggt agagcgcgtgttctaatttg 480 aacacgagta gaaaactttt ctaaaagcaa aaacgaaagg atgggtaaactgtattcgct 540 gaactgaata cgggcgactc tcctctaaat caaaattaag aaaggaattgaccccaccct 600 aaaagtagtg ggaaaaagat agttggtcta gcgagcatcg ctcactgcgcccaactccta 660 ttttcccttc gctttttgat gggtttggta tctttctcaa tataaaatataaataagaag 720 atagagcgtg tgttttgatt tgaacacgag cggaaaactc ggaaaatagataatctgact 780 gaaaaatcag gatttctcgt caggttccta attttcagtc gttttcttctcgctctttgt 840 atcataaatt atgtctatcc atattgctgc tcagcagggt gaaattgctgataaaattct 900 tcttcctggg gatcctcttc gtgctaagtt tattgcggag aatttccttgatgatgctgt 960 ttgttttaac gaagtgcgta acatgtttgg ttacactggt acttacaagggtcactgtgt 1020 atctgtcatg ggaactggga tgggaatgcc atctatttcg atttatgcgcgtgagttaat 1080 cgtagactac ggtgtgaaga aattgattcg tgtgggaact gcag 1124 45829 DNA Artificial sequence Synthetic probe 4 ttcatgcacc aatatattataataatcttc atccaataat aaggctgata aactagcatc 60 ttggtcaaaa tgtaagggaatttctcggcc gtccttcttt aatgtatatt ctggtaagat 120 actaaatagt tcaatcatagaaggaaactc aggattagtt gttgtaaatc gaaaaagctg 180 cgctttctca tctttcctgacttccttgat actctcccaa ttctaaaaaa tgattcaagg 240 tagtataaaa ttccttattttttacttcat caatgatgac catatcataa tcttttgttg 300 tgcgactttt aaatccttgcgaatccaata cgatagaggt agcagttccc ccaatcagaa 360 cataatagtt ctgaaaatccgcaaacgttt cttgaaaaat aactttcgta ttagctggca 420 tcatcttctc ccagatactgtaatatcata ttttctagtg cttcactctc ttcctctata 480 cgtgggtcat catcatcttttaaggtcaaa taaagagaaa tcggatctac aaattgttta 540 tcatgattat ttttaaaatcattccaaaac tcagatacaa aaggacgata tttccatatc 600 tctagcatct ttccttttaaaatatgctga gaaagtggca aggataactg attgaatttt 660 ctctgccata tgacatagctagtattttca tccgtttcag ctaaaaaagt tgaatgcgac 720 aaagcataag caccaccatatagaaggtta gaaacagatt ttatctgctt tatatcgcca 780 tctggcaata aaatccgttttttgatggga ttaaataaac atgacacgga ttttaagaat 840 aattctttct ttgacaccgtatatgtgtaa agcttatttt gcttgtttaa ccaatataaa 900 gctttaaaag tcctcaaacacctataaatt gttgagtttg gaagtccagt gacttgtgaa 960 agcaaatcaa catctactactttttgacct tttgtcaata aaaaggcaat ccacgttaat 1020 tgttcgctag gtgttaattccttagggact tcagtatcat tcgcattgag tactagtccc 1080 aatggaggga agaagaggtttcccttaaag tctacaaacg gaactctagc ttgaagtaat 1140 tgcttttttt cactgtctgataacttcgaa aacaccaaaa caacatccat attggctttt 1200 tcacccatag tgcgagcctgagtaacaaat gaactcaaac tcccccttct cttttcttta 1260 atcaataaaa atgattccttatgaaaagtc atttgagtat actcaaacct ttttacaaaa 1320 ctgattgata aagtcaaagtcagttcctca atactgacat caacataaaa ggattgaaaa 1380 attgacatta tctttttgacattcatgttt tttccttatt ctatcatttt tattatattt 1440 tatcattcgg tataataaaatataagtttt ttgataaaat aaccaaaaac tactccctat 1500 cccctctcac actaccctacatatcgtttg acatgcgact gatagttcag gaaaacttca 1560 aggagaactt ttctctcatccactatgcag gacttactat ttcacttcta ctcctatagg 1620 ctcaatttga cacttttctttttcgaattg ctcatatgcc tcctgaatag tgagttcgat 1680 ttatccaaat ttatttttgtctattttgat gaatacttcc acgaagatag tctgaaaatg 1740 aatttgcacc aattttccttctctttttaa gcatattatt ccagatatga ttgccctctt 1800 gttcagtaat aaattacgctttaaacgctt caatcagtat atctcctgtt gtcatatgtt 1860 ctaaagaaaa ttcttctacatatgatttaa catctcttag gttattactt cctaatatcc 1920 cattatgctt tttcgctaaggaaatagaag ccgcttctcc cttaccaata atcttgttac 1980 tatcatgatt tcttgttaaatctctatata atgcgtattc ttcagttcca atgtctatgc 2040 tcacaatctc agctgaacccttagctacca actgatctat cctagatttt aaatggggaa 2100 ttgtaggtat attgatttcatcatacacct cttgtggaat aacaatttta cccgaataga 2160 gcttttctaa aagatgttcagtaccaaccc ataaaaatac tgaaatacaa tctgcatcaa 2220 taaatacacg actagtcaaacgacaactcc cccctcttca tctagcccat atacaatatc 2280 atatctgaaa gcatctagtaacagttcctc atacttccct tgcgaaattc tgctattttc 2340 taaagtgttc agtcaattatatatatacta ataccatttc ctttttactt tctgacaaag 2400 gacgatataa acttgtatcatagcctaatc ttgaagctgt ctctataaca ctaatatcca 2460 tatttttaat ttcttctgcatcaaggtatc catcattcct caatctatat aacatagctt 2520 tatgactgat accataaaactgacccaatt ttataatatc ttctacttca agatgagttc 2580 tattggcatt ttctctgatttcctcaacca tcctatacag tgaaatggga aaattaaaaa 2640 ataagaagca aactgatccgcttttctttc agtttcatct ccttcaccaa tcaagataag 2700 actgactgaa ctcttcttcacctcatcata ataaagatga tacagttcat gtgctaaaga 2760 aaatctttac cttcctaatggcatgtctga attgactgca atgagactga aatgagttcc 2820 tttataacag accccgctaatattctttcc gagtccataa aataccagcg tcaaattttc 2880 tatcttttgt accaatttaaaaatatctat cggcgattca ccatcagctc ccaacttttt 2940 tctaagattt ggagctttattacttaaatc cattcgtgat attcctttca cctttttcca 3000 ttttggcaat catgctttgattcaaagaca aatactcagc aatcttatct tgagtaagat 3060 ttgaagatat tctaagttcttttattcttt ttccaacatc acatacattt atcatataca 3120 tttatcatat tcatttatcatattcataca cctctcataa aaagaatagc acactcttgt 3180 cataattttt taaataaaaaaattatgaca aaacaaggaa gcaatttatt gatgctgctt 3240 aaaaatctaa aattgatgatattaaaccta tttgatgaat tcctatcaaa aatcgtatct 3300 tcaacctcaa aacagtacttaaagctatcc gactcggttt acattgtcaa atttagattt 3360 tatttgagca taactttctagtttgctttt tgatttttgt ttaatatagt agcaaaaatt 3420 gaaggaaata tctccacaagaaaacgcata ctattaagct ttttcaagac ctaataatat 3480 gcgctgttct gatttgaaagacattccatt attattttac tgtaatcaag ccatctggct 3540 ctactgtgaa ttctggcttgtctgccagtg ttccgtctgg tttgaggtag taccagcctg 3600 ttccgtccgc tgactggataaaggcatttg ataccatggc gccttcttta gcgtctaagt 3660 agtaccaagt gtccttgtacttgacccagc ctgtcttcat ggcaccttct tcgttgaaat 3720 agtaccactt atcagcgattttcttccagc ctgtagccat ttcgcctgag ttgtcgaacc 3780 agtaccagtt gccgtctgtgtgcttcctcc agcggtctgc aagcatatag cctgaactgt 3840 caaagtagta ccaagtgccattgattttct caaacttgtc ttttggataa gagccgtctg 3900 agtgtacgta ccagtagccagtgtcattct cctgccagcc tgtttcaatc gtcaagccgt 3960 tctcaatatc atgcttaaactgctcacggc taatgcccca ttttgccaag taagggtatg 4020 gatccacatg gtctgagtggttgtttggtt ggttattcgt gcaatactcg tgcgttttaa 4080 ttccagctaa actccctgtatcaagcgttt tcggcaaacc tgcttcatct gctagattgc 4140 gtaagagttc gatataaaggcggtagtccg tcatgaactc ttctttagtt gaatggcttt 4200 caatcagttc aaccgctgcataggtctcag cattccaacc gcccccaacg tcccaggcac 4260 cattatcaac aggtcctacctgcatgatgc aaccgttccc aacaatgtgc gagaaaaaac 4320 taattctggg tctttccgccagtgataatc cgcttcattc tgtacggttg aatgcggatt 4380 cccagttgag tgtgcgtgtacttgcctata tggttgcacg ccgacttgag gcaaatctgt 4440 tcttaattta ctcacattaatttccatatt ctactcctta tcaattaaaa caactcattt 4500 tttacaatcc aaaaccagaaactcctttat ttctacctta caaagaagac aatcttagtc 4560 acgattaggc ttgtagatagaacctcaaaa cgcactattt tgacactgta aataggactg 4620 acaaggtctg cattctatctacaataacac cccagactaa aaagcttttc aaaagtatat 4680 ttttacagtc tctatatgtccttttcataa atactatact ttattatatc atataaaaga 4740 agtcaaaagt ctgttaaactattttcaaca ccaaactaaa gaagagaaca caagtttttt 4800 cgatgttcac tagaggaaatggattttatt cagtaaatcc aactaggatt gcactttggt 4860 tgccaaaatt gcctttccttcttttatcaa gggatgacgg aatagtgaga agtacagttg 4920 aactgtcatg gcaacccagataagaggttc acaaaggata acacccttat atcctgccca 4980 aggaataatc aaaaccacaaaaacgatttt tccgattagt tcaataaagc tagaaactag 5040 aggaaggatc ttctgccccaagccctgcaa gcaatgcgat aaatcaacaa gagctcaaaa 5100 tggataaaag gctgaactggatttgcagat agagacttcc attttctatc aagtaaccat 5160 ctgtcgaact agccaagaaggaaatcaagg ctgggctggc aaaaaagagg aaaatacaaa 5220 caaaaactgc ccaggatatacttaaacgac tgccgattcg aagaccttga gcaatgcggt 5280 caggtcgctt agctcctagattctgagaag caaaggtcgt cattgatgca gaaatagcgg 5340 tcataggaag aagggcaaaggtcataatgc gtcgagctgc cgtctgggca ctaataatca 5400 ctgcaccaaa tgtattaacagaagactgta aaatcacact gccgatagat acaattgaac 5460 tcatcaagcc catagccaaaccttgctcca agagatccgc gtacaagctt ttgtcccatt 5520 tgaaatgttt aaactgtggcaagagttctg gcacactttt acgaatataa taaaagcaga 5580 gaaccgctga taaaccttgtgaaataatgg tagcaagtcc tgcggattga actcccagat 5640 gcaattgcgt aataaaatagagatccagaa ccacattaac caaggcagag aaaatcagaa 5700 atcccagggc tgctagactgtcaccaatag accgcaacaa gcctgcaaaa agattataag 5760 caaagctgac acctacacaggtcacaatca tagaaatata ttgataagat tgaagaagaa 5820 tttctgcag 5829 5 3568DNA Artificial sequence Synthetic probe 5 ctgcaggata tttctgccttgtattgccag tggtttagcg ccacagccac atatctttta 60 cggtttttta ccgagaaaatcaggtcagca gaagcaattt ttttggcttg aaaaaagatt 120 atcctgaaac acagattttttatgaatcac ctcatcgtgt agcagacacg ttggaaaata 180 tgttagaagt ctacggtgaccgctccgttg tctctggtca gggaattgac caaaatctat 240 gaagaatacc aacgaggtactatctctgag ttattagaaa gcattgctga aacgccactc 300 aagggcgaat gtcttctcattgttgagggt gccagtcagg gtgtggagga aaaggacgag 360 gaagacttgt tcgtagaaattcaaacccgc atccagcaag gtgtgaagaa aaaccaagct 420 atcaagggaa gtcgctaagatttaccagtg gaataaaagt cagctctacg ctgcctacca 480 cgactgggga gaaaaacaataaagggagac aggatgtaat aattctgtct gtttctgttt 540 aacttaatta gtgatgataatataaagatg tatcacttgg tatagaagct ttggtattaa 600 gttttttatt aagcccatacggaataccga tggttggagc agcagttata gcgttcttag 660 aaggtataaa tagaaaaataaggtcatttt aaatcaaagg attgataaat cagaaagaag 720 gtgatttttt gcgaacatacgaaaataaag aagaactaaa agctgagata gagaaaacat 780 ttgagaaata tattttagaatttgataata ttccagaaaa tttaaaagat aagagagctg 840 atgaagttga cagaactccagcagaaaacc ttgcttatca ggttggttgg accaacttgg 900 ttcttaaatg ggaagaagatgaaagaaagg ggcttcaagt aaaaacacca tcggataaat 960 ttaaatggaa tcaacttggtgaattatatc agtggttcac agatacctac gctcatttat 1020 ctctgcaaga gttgaaagcaaaattaaatg aaaatattaa ttctatctct gcaatgattg 1080 attcgttgag tgaggaagaattatttgaac cgcatatgag aaagtgggct gatgaagcga 1140 ctaaaacagc gacttgggaagtgtataagt ttattcatgt aaatacggtt gcactttttg 1200 gaactttcag aactaaaatcagaaaatgga agaagatagt attataaatt atatttttaa 1260 ctttaaaaaa tttcataaaaatggttacca aaggcgatag aagaaaaact atcgtctttt 1320 tctttgcaaa tttttaagaaggggaggtga tcttgcatgg actttgaata tttttataac 1380 agagaagcgg aaagatttaacttcttaaaa gtaccggaga tattagttga tagagaagaa 1440 tttcggggct tatcagcagaagcaattatt cctttattcc atacttctta aacagacagg 1500 aatgtcattt aagaataactggatagacaa ggaaggcaga gtatttatct attttactgt 1560 cgaagaaatt atgaaaagaagaaatatctc aaagccaact gccataaaaa cattagatga 1620 gcttgatgta aaaaaaggaataggactgat cgaaagagta aggcttggac ttggtaagcc 1680 gaacatcatt tatgttaagactttatgagt atatttcagg taaaagaaaa tgacttacag 1740 aagtcaaaaa acttaacttcagaagtaaaa gattttaacc tcagaagtaa agaaaatgaa 1800 cttcaagagg ttaagaaccttgactctaac tatatagaga ataataagag taagtatagt 1860 aagagagaat atagttttggtgaaaacgga cttggaacat ttcaaaatgt gtttttagct 1920 gctgaagata tatcggatttacaaatcata atgaactcac agcttgagaa ttacattaga 1980 ctttctgcaa aactagaatcctagttcatg attgataata ccagcaatca aattcattcg 2040 taatccgaag cgtttacgatgattttgata ggttgttgaa aacattttaa acgtttttac 2100 tttggcaaag atgttctcaaccttgcttct ctccttagat agcgcatggt tacaggcttt 2160 atcttcaact gttagcggcttgagtttgct ggatttacgt gaagtttgtg cttgaggata 2220 tatcttcatg agcccttgataaccactgtc agccaagatt ttaccagctt gtccgatatt 2280 tctgcgactc attttgaacaacttccatat catgacaata gttcacagtg atatccaaag 2340 aaacaattct cccttgacttgtgacaatcg cttgagtctt catagcgtga aatttctttt 2400 taccagaatc attcgctaattcttttttta gggcgattga tttttacttc cgtcgcatca 2460 atcattaccg tgtcctcagagctgagagga gttcttgaaa tcgtaacacc actttgaaca 2520 agagttactt caacccattggctccgacgg agtaaagttg ctttcgtgaa taccaaaatc 2580 agccgcaatt tcttcataagttcgatattc tcgcacatat tgaagagtag ccataagaag 2640 gtcttctagg cttaatttaggttttcgtcc accttttgcg tgtttaagtt gataagctgt 2700 ttttaataca gctaacatctcttcaaaagt cgtgcgctga acaccaacaa aacgcttaaa 2760 tcgtgcatca gttagttgtttacttgcttc atcattcata gaactactat accatatttt 2820 gtttcgcagg aagtctattggaaagtaaga aatattgaag ctgaggctat tagaagaaat 2880 tgtgagcgtg gtgctattttttcaggtaaa ataaaatatc acgaagattc acagtttaaa 2940 ggagatcact atgttgaatgttatgctgtt ttagataata cggttatagc aagagataga 3000 ataacagtcc ctatcgatccgttatgtgga aaagatttta tagagtagca tataattgat 3060 tcttaactgg aatactcactatctctttac atcaagaaaa tgactaaaca gggaagtttg 3120 ccttcttccc tttttttgttatactagtag aagaaaaaat agaaagattt gtgggagtga 3180 aaagtcctgt ggactttttcagcctgagcc aagaaactcg aaagctcgta agtctgattg 3240 gctttttcaa tgtgaatcttaacttcatac tcccaaagag gtattagtgt cgtgtctcaa 3300 tcttatatca atgttatcggtgctggtttg gcaggttctg aagcagccta cccaaaatcg 3360 cagagcgtgg gtattccagttaaactttat gaaatgcgtg gtgtcaagtc aacacctcag 3420 cacaaaacag acaattttgctgagttggtt tgttccaatt ccttgcgtgg agatgatttg 3480 acaaatgctg ttggcctccttaaggaagaa atgcgtcgct tgggttctgt gattttggaa 3540 tctgctgagg ctacacgtgttcctgcag 3568 6 4528 DNA Artificial sequence Synthetic probe 6ctgcagattg gtatttatcg ccaccatctc cgtaagtctt agtacctgta aggacaattt 60tcttagcctt aattgtctta ccaaaatcaa tgtcttttgg cttattgtta tctggccaat 120cagttgcagt aaaggtatgc tccttgccag actcatctgt cacaacaagt ttcacatcac 180gcaagttacc atttgaacct gatccacgtg gaacataacg aagtccagtg atttcagttg 240cttctttcaa gaccatggtt gcaggcttgc ctacatctcc tccgccccat gatgtatgcc 300ataaactaga taagtttcca tcaaaggcat ttgcaagacc ttcttgagcc tgagcaggag 360ctgtaagact tgcaaagtca tctgctacca aagccgtttt cttctgaacc aaagcattct 420tcaaggcttc aatcttggca atctctgcac gcgcttcttc cacactgata tcatcatcgg 480cctgactgag gttaaagacc gcctctttca aagcatccat agactctttg gtgtagttag 540tcatggcaac cgttggcaag tagttcttca gagcattttc tgtcaacatc ttacctgtta 600gggtaatttc ttcgatttga agattatcca tcatgaagtc gttataacca cggaagttgg 660catttccacc agaatcacca cgagtattac ttgcatttcc agttgagtag atacctaccc 720aagtatcccc tgtttctgca cctgtcacga ggaaggttgc cttcttggct ttcttagaat 780ctgtccaagt atttggcaat tcatgcattt ccaagttgct tgcttgagta ccacgacgac 840ctgactggaa ttctccctta ccgactacaa aagcataggt attgtctgaa cctgcttcgt 900attcaaaggt tacacggtag gtcttacctg cttcaaaacg gaagttttgc ggaatagttt 960ggtaaaccaa gttacgacgg ctcactagtc catttgtctt gagtgaccaa tttccttcga 1020taacatcatc gactttctta ccattccaac cacgttgtgt atatggatcg tgtttttcag 1080acaagtgagt gcggttgtct tcgacacctt cgacaccacc cactacaaat gggaagatac 1140cctgagcaac attttcaaag tcttgcttga aggtgccttt acctgtatca tgcttgtctc 1200cgtacatgct tgaattgttt tcaaaggtac gaatttcatc aaagtaagtt gcttcatcac 1260cagcttcacg actcaatgtc agagtaacat ttgatacgtc cgatccagtt gtaaagaagg 1320cgtacatgtt ttggaagtaa cttgtatcgt caactgtagc attgttacga cgtgtattgt 1380gggcataggc ttttacatag ttgagggcga gagacttatt ggtataagta gtcacttctt 1440tttcaccagt atttacagtg atgctcgcct tggcattact acggttatcg acaccgacat 1500aaacggcata cttggtattt ggtttcaagc cagttaattt ctgagtgaga ctaacttttt 1560ctttgtttcc ttgaatacga agcatatcgt ttgccccttg agacttgaca atttctgcct 1620tagaagcatc gcctgaaatg gtccaatgtt tcaaggtacc actgttaaat ccttggtcat 1680agatgtgcat gccttcactc catgacattt caggattggt ttgtttcgaa cgatagagaa 1740cgtatggttg atttgctaga agatctaggg taattttacc atcttttaca gttagttctt 1800gctcttctgt cttaccttgg tcagttagct tgtaaaggta aaccttgctc tttgcccaat 1860cgcttggaag ggtccaagtt gttgcaccgg cctgcgtatt gaagtagtac atcttttcct 1920tatcagtaga aagtttctta ccatttgcat cccagttcca aggagtcaag taagctgaac 1980catcttggat gacacgtccg ttgagagtta ctgtacgttc gcgatattgt ggactattga 2040catcatttga cttacgagtt acaactactt tattattgtc agcatctacc aattccactc 2100gcatttctgg agtccattta taggtgctac cgttatcggt catagtccac cggtgtacca 2160ttttcccatt tacttacagt gaagtgttgg aagtacttgg tcatgacgtc atgggcaaat 2220aagttagtta catagccatt gtagtcactt cttccttgcc agccttcaaa gtctttcatg 2280ctgtagccac ctagcagtgg atagttggct gcaccaccat aacttctgta gtcccctacc 2340caagcatctt tttggtggtt acgtataaag cgggtgatgg cactgttgat acctttattg 2400gtgtagccac cgtaggtcaa gtcagctgcc cagtgatgga aggtagagtc gtactcacca 2460ccatggcccc actcgatcgc aaagcgccag ccttgtttgt taatttcttt agcaagaacg 2520tgggtagccc aggcaccgtt atcacctgat tgaccattac cccaaacgtc cacatagata 2580aagtcgagac cgtcaccaag ttttttcttc aaatcttccc aacgtgccaa acgaccatga 2640gctaggtcat aggcagcatc aatgttgata ccttgatcta gccagttcca accatagcta 2700tagcttccat ctggattctt acggagaatt ttttcattga agtatttaga ctcaggataa 2760gtttctgaag cgttaacgtg gatacctaga tgagctccat atttcttagc cttctcaatt 2820agggtcttga agtcttcgac accaccgata cgcttaccaa tatcagcata gttcaagtga 2880ccagagtcat ggccttcgct accatatcct ttaaggagaa caccttgccc aagaccatct 2940gtgtggagat tgattttctt gataccatcc aaggtcataa ggaatgggtt ttgtgcttga 3000gaaccaaagt tcatcgcgat acggtaagct gtgatatcct taactttttt ccaaccttga 3060ggattgttca taatgctacg ataagcaatg gcaccatcct gccaatcgac tttcttgtct 3120gcattggcat cttcagtgat aacaacctta gcacttggaa gttccttcgt gtattctggg 3180aaaacaatgc ccttataagc tttttcccat tgccattcag agctgtggat tcctacatag 3240ttggcatttc cgactgtttc tttataggct gtcaaacgag tccagtcatt cgaaccacca 3300ccatagctgt tttgagagtt actccaaaca ccagcagcaa gcttatctgt agaaacaaat 3360ccatacatgt aacccttggc tagatctttc attggattgg ttacatcgat atgatcatct 3420ccgctgacat gcgtattgtt tgacatggtt gccccatcaa acttagcacc agtttgatca 3480ctagaaacag agactaaagc attgccgagg aaactaatag aagaaagtag ttttctttcg 3540tcatcaatct tttgacctgg agtgacttga ttgtggttga caatcttggt cacatcaaag 3600tgcaattgat tgtccacaac ttgcaagcgt actgtcattt ccgcattgat taagtgagca 3660tcatcgcgaa gcttcatcaa gtactctgct gttgtctcat tgattttctt ataagtgact 3720tcaggggtga ttcggtggtt attgataaag acttggttga actgttgaac ctgtcctggc 3780aaagtatgtc cattcaagct gtattccttg acacgaggga aggcttggtc aatcactgct 3840ttgagaacct tagactgaat cgtgtcataa gtcaccttgc tatcatcaac ttcaggacct 3900gtttcttttt cagcaggggt atcctctgtt tttaccccct cttggttatc cgttttaacg 3960ctaacaactg ttcgctcatc gtcataagag cccgccttga gaagaatctt cttctcattt 4020ttaagatggt cattgaccgc agctggtaga gtcactgtgt caaagagatt gacatcgtta 4080ttgctggcat ttagctgacc gtctgacttg agagtgatag agagacggtt tgtgatctgt 4140ttcagagcag caacacgact acctctatac caagtgctag ttgttggaga tttatactcc 4200cagaaccagc catccttgtc ataaccgaca aaaacattat tcttggtatc tttaaatttc 4260aagaagacac caaagcgtga tttgcccttt tcagaatcat ctttgaaggt taaatcaaca 4320gttgcatttc cattggcatc aacggtcaag cccttctttt caaacagggc tggtttacct 4380gcgttatcat tttgagcagt tgaggataat tggttgtagc ggacaccttt ttcttctcgg 4440atagtgactg ttccctgttg ttcttttttc tctaccgttt gccattcagg ggttaccgtc 4500ttaggtgttt caggtttagc agctgcag 4528 7 5579 DNA Artificial sequenceSynthetic probe 7 ctgcaggctt ccctttagca gttacagcct gtttcttacg gtctttttcactccggccaa 60 tcggcgcttc aattacacca cgatcattag gcagatttcc atgaacaatcgcccaatatt 120 tgcggagaga ctttttatct ttgagttctt gggcaagtac tagatgcgcatcatcgtttt 180 tagcaatcat gagaagacct gacgtatcct tatcaatacg gtgaacaatccctggacgca 240 gaaccccatt gatacccgac aagtccttaa tatgatacat gagggcatttactagggttc 300 cactggtatg accagcactc gggtgcacaa ccattccctg aggtttgttaacgacagcca 360 catcctcatc ttggtagact atttctagcg gaagatcctc agccacatactctaatacct 420 ctggttctgg cacatggtaa gtgacgacat caccctcttg gactgtgtatttagctttct 480 tgacttgacc attgaccaag acctggcctg atttaatttg ttcattcgcgagactacgtg 540 ataattctga caaatctgac aaagccttat ccaaacgcag accaccagtttcaattttaa 600 tttccattta tttcctcttt tagcattgca atcaataaaa taatcactccaaccgtcaga 660 tagctatctg ccacattgaa aattgcaaag ttgataaagt caaggtggaacatatccaca 720 acaaagccct gactgaccct gtcaataaag tttccaagac cacccgcgattattagagtc 780 aaacccaaga ccatccagaa tgagtcctcc atgtgtttat gtaaataccaaatggcacct 840 atcacgacaa ccagagtaat gacagcgaat aacagctgct gatcttgtaagatagaaaag 900 gctgcacctc gattttgcag gtaggtcaag ctaacgaaat tgggaatccaggagcgcact 960 tcacccagtg gaatctgctg gacgatatag gatttgacca actgatccagcccaattaaa 1020 agcagtacaa tgactgccac tattgctctt tttttcatga tttcctcttttgatcaaaat 1080 attcttgcat gacttctacg aagagagtcc cagcttgact aagctccacttcttcacgtt 1140 taacatagac catgcggtta tctaggttat ccttgagacg aataactgtaatgccattaa 1200 cactgtcact atctaaaaat ccagaacctg tcgcataggc gtccgtccgctccaaaatac 1260 cattcaaggt ggcacggtct gtcacattaa acatctgtga gctagcgctggtatcgacaa 1320 agttctctga ataataaagg tactcgtctt tctcttgagt gaaacagaccgttggtaaat 1380 ccgctaaatc ctccatgact aattcctctt tctgggctaa aggatgaccctcacagagat 1440 aaatatgggt atggaaggaa tcaattcgat gacctccaga cctaacttttcaacccgttg 1500 cataatcccc tttttatttt gattgtggag gtagataatc ccaatctcactatgcccttg 1560 cgccacttca tctaatattt gaacagtagt tgattcaaaa atacggaagttcttatagtc 1620 aggatagcgc tctgaaaagg ccgtaatagt tggtggtaag aagtcatagtgctggctagc 1680 aacggaaaat tcatcttttt cttcttcagg attggcatac tgattttgaaaaatatcaaa 1740 tcctttaacc aattcttgcg ctttttcata aaattccatc ccacgacgggtcaagaaagt 1800 ccctgagctg gtccgacgga aaatcttaaa gcccaactct ttttccaaatcacgaacaga 1860 aatagacaga ctcggctgac taacatacat cttttcagca gcttcacgaaaagtaccact 1920 attggcaata gccacaacat agcgtaattg ttgaatgttc atcttctacccccaacttct 1980 ttatcttttc attataccat attttagaag ttttccaaaa aggaaaaaagaacatcctat 2040 tcttcttaac tatcttcact atctgccttt ctcacgccaa tcttattttcaaaatcaatt 2100 caaaaaaata agtggctaca caccacttca gtatcggaaa gaaaaacgttgactatttgt 2160 gaaaaaagaa aatgccggaa aattccgaca tttttttagt aagctaacttcctgaaaata 2220 ggagtaacca aacatcccag aaggcagtga tattgatgag aatatgaactagtattggat 2280 agtagagatt tttagtcatg cgatacaata gagctaaaat cagacctccactagcataaa 2340 tgaaaaaagc aagaggagtt aaagcaaaat tgatatgaat ataaccgaaaataatagcag 2400 aaagcaaaac atctccgtac caaggtgagt ttttgaaaaa ggttgtcataagcacacctc 2460 gataaatcaa ttcctcagca ataggggcga tgaagcaaac gatgagcaagaaataaggga 2520 actcctgtct ccccatcatt tctatcgttt cattcaaaga aatctgatttgaagacaggg 2580 atatgaaata cgaaaagagg aagtcagaca tatatgaaat gatgtagcccagtaaaaggt 2640 agatgaagta cctcagctgc cacttgaaat gaaaaatttt ctttcctgcaaaaaccagta 2700 gatagatgac cgctaagaaa agaactccgc tctccatcaa aagcagaatctgaaaaaatt 2760 cacgacttgc tggtagatag ggctttgcga ggtgattgaa aactctccaaaaccaagttg 2820 atttgtaaaa aattaaggac aatgctagta aaatttgaat agcccgttttttcatattaa 2880 attctctgct ttctcccctt gtttcttcat attactaata aattttacttaaaatctcgc 2940 agcacttcct ttgcaaagat gattgcctcc tccaatatat cctaaatcataggtgcctct 3000 gggacaaaat ccatgaaccg gcagagttaa tactggttct atttcccaatcgtacctacc 3060 attaggattt ttaccatatg aatctgctcc accttgtatc agctctaattcttcatttgt 3120 tagatttata ttttttgtca ttaaaatcat gatatatttc ctccattattcttcagatta 3180 gttgagtaat ctgatactca tacctactta caaaaaaact attatattaagttggttttt 3240 taatttgtct agttgcaaca ttgacaaaca tagtatagca tatctttgcgaaatatcctc 3300 ttcaaatcat gaattgtcat caaaacatct taaactataa aatcaattagtctcaagctt 3360 tctatcaatt tcttctccaa aatatgctat aataatagca aaagataaagaaggaagacc 3420 tatgattaaa ctactagcct tggatatgga cggcaccctc ctcaatgaagccaaggaaat 3480 cccacaagct cacattactg ctattcacaa agccattgaa aaaggtgtcaaactggttct 3540 ctgtacggtc gccccctttt cggtgttcct cccctactac aaaaaactgggactcgacct 3600 ccagaatgag tatgtgattg ttaacaacgg ttgttcaact caccagactagcgactgggg 3660 cttggttgac tggcaagaac ttagtccagc tgacatcgaa tacctctatgaccttgctga 3720 aaagagtgat gttcagttga cactttttga cgagtcacat tattttgttctcggtggaaa 3780 gcccaatcaa gttattgaaa atgatgctaa actagtattt tcagacctgactgaaatttc 3840 tcttgaggaa gcgactagtg gaaagttccg gatgttccaa ggtatgtttttaggaacaaa 3900 agaacaaaca gacgattttg agcagcgttt tgctgaagag ctttgccaacgattcagtga 3960 gttcgttcgc agcctgtcat ttatgaagca atgccacttg gaacgacaaaggctactgct 4020 cttcacgact agctgagatt ttgaagattg attcctcaga gattatggccatgggcgatg 4080 ctaataacga tatcgaaatg ctccagtttg cagggcttgg gattgcaatgggaaatgcca 4140 gcgattatgt caaatctctt gcggatgccg ttacctcaag caacgaagaagacggcgttg 4200 cgcgtgctat tgagaaatat attctataaa aaagaaaagc aaatagacagaagttactgc 4260 tatttgcttt tttgctaata ttttaaaata cggactaatc aatagagaagaatagcgaat 4320 cataaaaatc gatttactag atgccatagt atcttatagt tgctaataagaagttagtct 4380 agcagaatga atctctcatt tctcccacac taatctgtaa tttgtctatacattttctgg 4440 tcttctatct cgaaacaagc cccagtttag gcgttcactt gctcccttgtctaggttata 4500 agtagctaac agaacagctt cttcttgtct ttcagctcgt tctagaatagctcctgtttc 4560 atccgtcata aaggaggaac cgtagaagtc aagactggaa ctctgtccgccattttcctc 4620 actaggagta acctcctcta aaccataacg attggctgcg atgactggaacaatattcgc 4680 tgctgcgtgc ccttgcatag tacgttgcca gtgaccacaa ctatctgtatccaaaattgg 4740 ctctgaaccg atagctgtag gataaaagag caattcagca ccattcaatgcaagacagcg 4800 cgctgtttca gggaaccatt gatcccaaca gataccgata ccaatcttagcatagcgagt 4860 attccagacc ttgaaaccag tgttaccagg cgtgaaatag aatttttcttgataataatg 4920 gtcatctggt atatgggtct ttcgataaac gcccagcact tccccatctgcatcaatgac 4980 ggcaatagag ttatacaaga cattaccatc tttttcatag aaactgattggtaaaacaac 5040 ttgtagttcc ttagcaatca ccttaaaatg ctgaatggca gtattttccgctacagattg 5100 ggcatactgg tagtagtcat actgacgttc ctgacagaaa tagggatgttcaaacaactc 5160 gggcaagaga ataatttggg ctccttgctc agcagcctga cgtactaaacgctctgcggt 5220 ttggatattt gttgccacat ccttagcgca ttgcatctga atggttgcaactcttacatt 5280 tctcatcttt ttctcctatt ctggaatttg ttgggtgata cagtggatattgccaccacc 5340 taagagaata tctctggctg gtattccgac aactttacgg tctgggaaacacttgctgag 5400 gatatctaag gccacttggt cgtttacatc ctcaaactgt ggaaccaagacagccttgtt 5460 ggcgatataa aagtttacgt aggaagctgc tagtcgttca cctgcgtatcgctcttcttc 5520 tccttcttca tagatgtagc ctggcaaatc ttcttctgtc acaacttgtcgaactgcag 5579 8 24 DNA Artificial sequence Synthetic primer 8cgacgttgta aaacgacggc cagt 24 9 17 DNA Artificial sequence Syntheticprimer 9 caggaaacag ctatgac 17

What is claimed is:
 1. A purified nucleic acid probe for diagnosinginfectious diseases caused by Streptococcus pneumoniae, the nucleic acidconsisting of a nucleotide sequence selected from the group consistingof SEQ ID NOs: 1, 2, 5, 6, and 7, and nucleotide sequences fullycomplementary thereto.
 2. A purified nucleic acid probe according toclaim 1, the nucleic acid consisting of the nucleotide sequence setforth in SEQ ID NO: 1, or a nucleotide sequence fully complementarythereto.
 3. A purified nucleic acid probe according to claim 1, thenucleic acid consisting of the nucleotide sequence set forth in SEQ IDNO: 2, or a nucleotide sequence fully complementary thereto.
 4. Apurified nucleic acid probe according to claim 1, the nucleic acidconsisting of the nucleotide sequence set forth in SEQ ID NO: 5 or anucleotide sequence fully complementary thereto.
 5. A purified nucleicacid probe according to claim 1, the nucleic acid consisting of thenucleotide sequence set forth in SEQ ID NO: 6, or a nucleotide sequencefully complementary thereto.
 6. A purified nucleic acid probe accordingto claim 1, the nucleic acid consisting of the nucleotide sequence setforth in SEQ ID NO: 7, or a nucleotide sequence fully complementarythereto.
 7. A probe according to claim 1, said probe further comprisinga detectable label.
 8. A purified nucleic acid comprising of thenucleotide sequence set forth in SEQ ID NO: 4, or a nucleotide sequencefully complementary thereto, wherein said nucleic acid hybridizes togenomic DNA of Streptococcus pneumoniae, but fails to hybridize togenomic DNA of Streptococcus agalactiae, Streptococcus anginosus,Streptococcus constellatus, Streptococcus equisimilis, Streptococcusfaecium, Streptococcus faecalis, Streptococcus mitis, Streptococcusmorbillorum, Streptococcus pyogenes, Streptococcus sanguis, andStreptococcus salivarius, under the following hybridization and washingconditions: hybridization overnight at 42° C. in a hybridizationsolution comprising 45% formamide and 5×SSC; and washing twice for 20minutes at 55° C., in a washing solution comprising 0.1×SSC and 0.1%SDS.
 9. A purified polynucleotide consisting of a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and nucleotidesequences fully complementary thereto, wherein said polynucleotidehybridizes to genomic DNA of Streptococcus pneumoniae, but fails tohybridize to genomic DNA of Streptococcus agalactiae, Streptococcusanginosus, Streptococcus constellatus, Streptococcus equisimilis,Streptococcus faecium, Streptococcus faecalis, Streptococcus mitis,Streptococcus morbillorum, Streptococcus pyogenes, Streptococcussanguis, and Streptococcus salivarius, under the following hybridizationand washing conditions: hybridization overnight at 42° C. in ahybridization solution comprising 45% formamide and 5×SSC; and washingtwice for 20 minutes at 55° C., in a washing solution comprising 0.1×SSCand 0.1% SDS.
 10. A probe for the diagnosis of infectious disease causedby Streptococcus pneumoniae, consisting of the purified polynucleotideaccording to claim 9, said probe further comprising a detectable label.11. A vector containing a nucleic acid insert, wherein the nucleic acidinsert consists of a nucleic acid, nucleic acid probe, or polynucleotideaccording to any one of claims 1, 2, 3, 4, 5, 6, 8, or
 9. 12. A purifiednucleic acid probe for diagnosing infectious diseases caused byStreptococcus pneumoniae, wherein the nucleotide sequence of saidnucleic acid consists of a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and nucleotide sequencesfully complementary thereto.
 13. The nucleic acid probe according toclaim 12, wherein the probe further includes a detectable label attachedto the nucleic acid.
 14. The nucleic acid probe according to claim 12,wherein the nucleic acid contains at least one radioisotope.
 15. Avector containing a nucleic acid insert, wherein the nucleic acid insertconsists of a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 1, 2, 5, 6, and 7, and nucleotide sequences fullycomplementary thereto.